Process of preparing microspheres for sustained release having improved dispersibility and syringeability

ABSTRACT

Disclosed is a process of preparing sustained release microspheres, containing a biodegradable polymer as a carrier and a drug, using spray drying. The process comprises preparing a solution, suspension or emulsion containing a biodegradable polymer, a drug and a solvent; spray drying the solution, suspension or emulsion; and suspending spray-dried microspheres in an aqueous solution containing polyvinyl alcohol to remove the residual solvent and increase the hydrophilicity of the microsphere surface. The process enables the preparation of microspheres having high drug encapsulation efficiency, almost not having a toxicity problem due to the residual solvent, and having good syringeability. The microspheres prepared according to the present invention release an effective concentration of a drug in a sustained manner for a predetermined period when administered to the body, and are thus useful in the treatment of diseases.

TECHNICAL FIELD

The present invention relates to a process of preparing sustainedrelease microspheres comprising a biodegradable polymer as a carrier anda drug. Such microspheres are an injectable sustained-releaseformulation, which enables the sustained and uniform release of a drugso as to maintain its biological activity in the body upon subcutaneousor intramuscular injection.

BACKGROUND ART

A number of approaches have been used to encapsulate bioactive agentsinto microspheres of polymers for sustained release. Most of them arebased on phase separation (U.S. Pat. No. 4,673,595, EP 52,510),cryopulverization after melt extrusion (U.S. Pat. Nos. 5,134,122,5,192,741, 5,225,205, 5,431,348, 5,439,688, 5,445,832 and 5,776,885),double emulsion evaporation (w/o/w, water/oil/water) (U.S. Pat. Nos.4,652,441, 4,711,782, 4,954,298, 5,061,492, 5,330,767, 5,476,663,5,480,656, 5,611,971, 5,631,020 and 5,631,021), single emulsionevaporation (o/w, oil/water) (U.S. Pat. Nos. 4,389,330 and 5,945,126;Shameem M, Lee Hee Yong, DeLuca P. P., AAPS Pharmsci., 1 (3) article 7,1999; Kostanski J. W., Pharm. Dev. Tech. 5, 585-596, 2000), and spraydrying (IE920956).

Phase separation encapsulation is a process for preparing microspheresin which a biodegradable polymer is dissolved in an excessive amount ofan organic solvent, such as methylene chloride, and a drug dissolved ina small amount of water is added to the polymer solution with stirring.Silicon oil is then added to the polymer-drug mixture at a constant rateto form embryonic microspheres, and an excessive amount of anon-solvent, such as trichlorofluoromethane, is added to the solution toextract the organic solvent from the embryonic microspheres. Thesolidified microspheres are recovered by filtration, and dried underpressure. However, the phase separation method is problematic asfollows. Since the toxic solvent such as methylenechloride is notsufficiently removed by drying under pressure, the residual solventreduces the stability of formulations, and may also be detrimental tohealth when administered to the body. Also, the excessive use ofnon-solvent, such as freon, hexane, heptane, cyclohexane, andtrichlorofluoromethane, for the solidification of embryonic microspheresis not cost-effective upon mass production and causes seriousenvironmental contamination.

By contrast, cryopulverization after melt extrusion permits minimal useof toxic solvents. This method is a process for preparing microspheresin which a mixture of a biodegradable polymer and a drug ismelt-extruded through an extruder at a high temperature and pulverizedat a low temperature. The biodegradable polymer-drug mixture may beobtained by homogeneously mixing a polymer and a drug in a solvent, suchas methylene chloride, with an agitator, and removing the organicsolvent using a rotary evaporator or a vacuum dryer, or by cryo-millingat a low temperature and sieving each powder and blending the two finepowders. The latter case does not have the problem of the residual toxicsolvent because it does not employ a toxic solvent during microspherepreparation. However, the procedure for preparing microparticles doesnot exclude the possibility of an interaction between the polymer andthe drug and denaturation of the drug due to high temperature and highpressure upon melt-extrusion, and denaturation of the drug due to heatlocally generated during cryopulverization. This method is alsodifficult to use to make microspheres having a uniform size, which arethus easy to inject.

The two methods for preparing microspheres, in addition to the problemsof residual solvent, difficulty in mass production and drugdenaturation, have another disadvantage in that a biodegradable polymerused for the sustainable release of a drug is non-hydrophilic and thuspoorly dispersible in an aqueous suspension for injection.

Water-in-oil-in-water (w/o/w) double emulsion evaporation has commonlybeen applied to encapsulate hydrophilic drugs, such as peptides orproteins, into polymeric microspheres. In this W/O/W method, ahydrophilic drug is dissolved in water, and this aqueous phase isdispersed in an organic phase containing a biodegradable polymer toyield a primary emulsion (water in oil). This primary emulsion is againdispersed in a secondary aqueous phase containing an emulsifier. Singleemulsion evaporation (oil in water (o/w)) has been commonly used in theencapsulation of lipophilic drugs. In this O/W method, a drug and abiodegradable polymer are co-dissolved in a mixture of suitable organicsolvents (e.g., methanol and methylene chloride), and the resultingsolution is dispersed in an aqueous phase. In both emulsion evaporationmethods, as an organic solvent is removed by extraction or evaporationduring polymer dispersion in an aqueous phase, the polymer decreases insolubility and is thus solidified to form microspheres. In thesemethods, the technically important factor is the encapsulationefficiency of bioactive drugs.

Most hydrophilic drugs leak in large amounts when dispersed in anaqueous phase, resulting in low encapsulation efficiency. To solve thisproblem, Okada et al. employed material such as gelatin in themicrosphere preparation based on double emulsion evaporation. Thismaterial increased the viscosity of a primary emulsion and decreased thediffusion rate of a drug (an LHRH derivative) into a secondary emulsion,resulting in enhanced drug encapsulation (Okada, H. and Toguchi, H.,Crit. Rev. Ther. Drug Carrier Syst., 12, 1-99, 1995). Similarly, thesingle emulsion evaporation method also can enhance drug encapsulationby suitably increasing the concentration of a biodegradable polymer(PLGA) dissolved in an organic solvent phase. Typically, microspheresprepared by double emulsion evaporation are more porous than thoseprepared by single emulsion evaporation, and thus have increased surfaceareas, leading to a relatively high initial release rate of a drug.

However, the single and double emulsion evaporation methods forpreparing microspheres, like the phase separation method, have thefollowing disadvantages: difficulty in the removal of an organic solventused for dissolving a biodegradable polymer, difficulty in massproduction procedures due to changes in solvent removal rate, allergicreactions to gelatin used for increasing the viscosity of a primaryemulsion, the possibility of a drug becoming denatured and losing itsactivity due to strong shearing force applied for making smallmicrospheres during primary emulsion preparation, limited drugencapsulation, and the like.

The spray drying method has also been used for preparing finely atomizedparticles. In this method, typically, a solution of a material to bedried, or a suspension or emulsion in which a biodegradable polymer anda drug are homogenously dissolved, is supplied to a nozzle, sprayedthrough the nozzle, and exposed to heated air to evaporate the solventused. In particular, in the case of preparing sustained releasemicrospheres, the drug release rates of prepared microspheres greatlydepend on the composition or content of a biodegradable polymer, thetype or content of an additive, the composition of a solvent, and thelike. In addition to the above processing parameters, other parametersaffecting the morphology, size or properties of microspheres may beemployed to control the release rates of drugs, the parameters includingthe type of a spray nozzle through which a spray solution is sprayed(for example, a type that atomizes droplets using compressed air, a typethat atomizes droplets using centrifugal force when a spray solutionflows into a disc rotating at a high speed, a type that atomizesdroplets using ultrasonic waves generated when a vibrator vibrates,etc.), supply rate of a spray solution, and temperature, supplied amountand supply rate of dry air. In addition, the spray drying method, unlikeother methods for preparing sustained release microspheres, isadvantageous in that it provides a continuous process, which facilitatesmicrosphere production and thus conversion from small-scale tolarge-scale production.

Although the spray drying method has the advantage of permittinglarge-scale production of microspheres, it has disadvantages as follows.The solvent used is not sufficiently removed merely by spray drying. Theresidual solvent causes a problem in the stability of the biodegradablepolymer upon long-term storage, leading to changes in drug releaseprofiles of microspheres. Another disadvantage of this method is thatsince biodegradable polymers used for drug encapsulation are mostlynon-hydrophilic, microspheres prepared are not suspended well and arethus difficult to accurately administer.

As described above, most conventional methods of preparing sustainedrelease microspheres employ a toxic solvent, and have problems includingresidue of the toxic solvent used, the microsphere size not beingsuitable for injection, poor suspendability of microspheres, anddifficult mass production.

The present inventors intended to provide a process for preparingbioactive drug-loaded biodegradable polymer microspheres, which are easyfor mass production, by solving the aforementioned problems.

DISCLOSURE OF THE INVENTION

It is therefore an object of the present invention to provide a processfor preparing sustained release microspheres comprising a biodegradablepolymer as a carrier and a bioactive drug, the microspheres being easyfor mass production, not containing a residual toxic solvent, which is aproblem of conventional methods of preparing sustained releasemicrospheres, having high drug encapsulation efficiency, and having auniform size suitable for injection.

The present inventors conducted intensive studies, and found that whenmicrospheres, which are obtained by spray drying a solution, suspensionor emulsion containing a biodegradable polymer, a drug and a solvent,are suspended in an aqueous solution containing polyvinyl alcohol toremove the residual solvent, the microspheres have improvedsuspendability and syringeability and high drug encapsulation efficiencywith no residual toxic solvent, thereby leading to the presentinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and other advantages of thepresent invention will be more clearly understood from the followingdetailed description taken in conjunction with the accompanyingdrawings, in which:

FIGS. 1 a and 1 b show the dispersion/withdrawal rates andsyringeability of microspheres prepared in Preparation Example 1according to the present invention in an aqueous solution, wherein,after a test was carried out according to the method described in TestExample 1, the withdrawal rates of microspheres in each tube (FIG. 1 a)and the deviation in each tube for the mean withdrawal rate (FIG. 1 b)were measured;

FIG. 2 shows the serum testosterone concentrations in male SD rats (n=5)which were subcutaneously injected as described for Test Example 5 witha single dosage of leuprolelin-loaded microspheres prepared inPreparation Example 8; and

FIG. 3 shows the serum octreotide concentrations in male SD rats (n=6)which were subcutaneously injected as described for Test Example 6 witha single dosage of octreotide-loaded microspheres prepared inPreparation Example 9.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention relates to a process of preparing sustainedrelease microspheres, in which a solution, suspension or emulsioncontaining a biodegradable polymer, a drug and a solvent is spray dried,and microspheres thus obtained are dispersed in an aqueous solutioncontaining polyvinyl alcohol, thereby more easily removing residualsolvent and improving suspendability of the microspheres uponadministration.

In detail, the present invention relates to a process of preparingsustained release microspheres having high drug encapsulationefficiency, almost no residual solvent, and improved suspendability bydissolving and spray drying a biodegradable polymer and a drug,suspending microspheres thus obtained in an aqueous solution in whichpolyvinyl alcohol is dissolved, and recovering, washing and freezedrying the microspheres.

In one aspect, the present invention provides a process of preparingsustained release microspheres, comprising spraying a solution,suspension or emulsion containing a biodegradable polymer, a drug and asolvent into a dry chamber and drying it using dry air to remove thesolvent; and dispersing spray-dried microspheres in an aqueous solutioncontaining polyvinyl alcohol to remove the residual solvent and improvedispersibility of the microspheres.

The term “biodegradable polymer,” as used herein, refers to a polymerthat slowly degrades when administered to the body, and is thus notharmful to the body. Examples of such polymers include polylactide(PLA), polyglycolide (PGA) and their copolymer,poly(lactide-co-glycolide) (PLGA), polyorthoester, polyanhydride,polyhydroxybutyric acid, polycaprolactone, polyalkylcarbonate, andderivatives thereof.

The term “drug,” as used herein, includes peptides having biologicalactivities, such as anticancer agents, antibiotics, antipyretics,analgesics, anti-inflammatory agents, antitussive expectorants,sedatives, antiulcer agents, antidepressants, antiallergenic agents,antidiabetic agents, antihyperlipidemic agents, antituberculous agents,hormonal agents, bone metabolic agents, immunoinhibitors, angiogenesisinhibitors, contraceptives, and vitamin-like agents. In particular,biologically active peptide or protein drugs are preferred. Examples ofoligopeptides having biological activities include insulin, somatostatinand derivatives thereof, growth hormone, prolactin, adrenocorticotropichormone, melanocyte-stimulating hormone, thyrotropin-releasing hormoneand salts and derivatives thereof, thyroid-stimulating hormone,luteinizing hormone, follicle-stimulating hormone, vasopressin andderivatives thereof, oxytocin, calcitonin, parathyroid hormone,glucagon, gastrin, secretin, pancreozymin, cholecystokinin, angiotensin,human placental lactogen, human chorionic gonadotropin, and enkephalinand derivatives thereof. Examples of polypeptides include endorphin,interferon (α-type, β-type, γ-type), interleukin, tuftsin, thymopoietin,thymosin, thymostimulin, thymic humoral factor (THF), serum thymicfactor and derivatives thereof, tumor necrosis factor, colonystimulating factor (CSF), motilin, dynorphin, bombesin, neurotensin,bradykinin, caerulein, urokinase, asparaginase, kallikrein, substance P,nerve growth factor, blood coagulation factor VIII and IX, lysozyme,polymyxin B, colistin, gramicidin, bacitracin, proteinsynthesis-stimulating peptide, vasoactive intestinal polypeptide,platelet-derived growth factor, growth hormone-releasing factor, bonemorphogenetic protein, epidermal growth factor, and erythropoietin.

In the present invention, the aqueous solution containing polyvinylalcohol is used to more effectively remove the residual solvent insidemicrospheres immediately after spray drying and to disperse microsphereswell in an injection solution upon administration. This aqueous solutionis removed through an additional washing step, and finally remains inmicrospheres at an amount less than 1%. Also, when microspheres aresuspended in the aqueous solution of polyvinyl alcohol to remove theresidual solvent, the removal speed of residual solvent may becontrolled by altering the temperature of the suspension. Using thisfeature, upon small-scale production, the residual solvent may beremoved within a short time at room or ambient temperature. Uponlarge-scale production, the suspension is maintained at low temperatureto remove the residual solvent at a slow speed, thereby preventingdeterioration of products due to extended handling time of largequantities of products.

In an additional dispersal step, the content of polyvinyl alcohol in theaqueous solution is preferably 0.01-20% (w/v), and more preferably0.05-10% (w/v). Polyvinyl alcohol has a molecular weight of 3,000 to300,000, preferably 5,000 to 100,000, and has a hydration rate of 75% to95%. The amount of polyvinyl alcohol remaining on the surface ofmicrospheres is preferably 0.02-1% (w/v), and more preferably 0.05-0.5%(w/v).

The term “solvent,” as used herein, refers to a material that is able todissolve a biodegradable polymer and/or a drug. An appropriate solventmay be selected by those having ordinary skill in the art according tothe type of biodegradable polymer. Glacial acetic acid is preferred.

The present invention includes a step of dispersing microspheres in anaqueous solution of polyvinyl alcohol to improve the dispersibility ofmicrospheres. The dispersal step is carried out for about one minute orlonger to achieve maximal effects. The preferred dispersal time is 5 minor longer.

In a patent application filed prior to the present invention (KoreanPat. Application 10-2003-0023130), the present inventors improved drugencapsulation efficiency and release profiles of microspheres byhomogenously dissolving a biodegradable polymer and a drug using anon-toxic solvent and spray drying the resulting solution, and suggesteda spray drying method facilitating the mass production of microspheres.This spray drying method has advantages including easy mass production,low residual solvent, high drug encapsulation efficiency, and ideal drugrelease profiles, but has drawbacks including difficult dispersion andsuspension of sustained release microspheres obtained by spray drying inan injection solution due to non-hydrophilicity of a polymer containedin the microspheres, and requirement of an additional step for removingthe residual solvent to ensure the stability of microspheres uponlong-term storage. Thus, the present inventors intended to solve thesedisadvantages in the present invention.

A conventional method of preparing microspheres by spray-drying usingtwo nozzles is disclosed in U.S. Pat. No. 5,622,657. To improve thedispersibility of polymeric microspheres, which are apt to adhere toeach other or aggregate when prepared by spray drying, the citedinvention provides a method of coating drug-loaded polymericmicrospheres with an aggregation-preventing agent using two or morenozzles, in which a polymer solution containing a biologically activesubstance and an aqueous solution of an agent for preventing aggregationof microparticles are sprayed separately from different nozzles at thesame time. A similar method is disclosed in Korean Pat. No. 0177309, themethod being characterized by spraying a dispersion in which awater-soluble dispersing agent is dissolved in the direction opposite tothe spraying direction of a biodegradable polymer solution containing anactive ingredient and the flow direction of dry air in order to coat aportion or all of sustained release microparticles with thewater-soluble dispersing agent. The cited inventions intended to preventmicrospheres from aggregating by spraying an aqueous solution forpreventing microsphere aggregation immediately after microspheres areformed using spray drying, but there are following disadvantages. Sincemicrospheres are coated with an aggregation-preventing agent immediatelyafter being spray dried, a toxic solvent used for dissolving a polymerfor preparing microspheres remains in microspheres in large quantities.In addition, when microspheres are suspended in an injection solutionfor administration, an excess amount of a dispersing agent is used.Moreover, aggregation-preventing agents used in the cited inventions,such as hydroxypropylcellulose, carboxymethylcellulose, glycin, alanin,gelatin and collagen, do not improve the suspendability in an injectionsolution or the syringeability, resulting in non-uniform dosages.

On the other hand, in order to enhance the fluidity of granules fortablet preparation or drug particles and improve the solubility ofdrugs, a water-soluble dispersing agent, such ashydroxypropylmethylcellulose, hydroxypropylcellulose, and polyvinylalcohol, may be spray dried together with a drug to be contained in theresulting preparation in an amount of about 4 to 19 wt % (D. Ermis, A.Yuksel, Preparation of spray-dried microspheres of indomethacin andexamination of the effects of coating on dissolution rates, J.Microencapsulation, vol. 16, No. 3, 3,315-324(1999)). In the literature,the water-soluble dispersing agents are used to enhance the solubilityof poorly water-soluble drugs, thereby dissolving the drugs withinseveral hours, and increase the fluidity of drug particles, therebyfacilitating processing such as tabletting. Polyvinyl alcohol has beenregistered as a material liable to induce cancer when parenterallyadministered to animals, and thus the residual amount thereof should becontrolled (Carcinogenic Studies on Water-Soluble and InsolubleMacromolecules, Archives of Pathology, 67, 589-617, 1959). The presentinventors found that when polyvinyl alcohol, suspended in a polymersolution, is sprayed as described in the above literature, the contentof polyvinyl alcohol, which is difficult to degrade in the body,increases, and the initial drug release greatly increases. Such a highinitial drug release may cause side effects, and may lead to a reduceddrug release duration, thereby not guaranteeing suitable therapeuticeffects.

The present inventors prepared microspheres in which the residualsolvent is additionally removed and which have improved syringeability,through a process comprising preparing microspheres using a spray dryingmethod developed by the present inventors prior to the presentinvention, suspending the microspheres in an aqueous solution containingpolyvinyl alcohol, and washing and recovering the microspheres.

The process of preparing biodegradable polymer microspheres containing adrug having biological activity according to the present inventioncomprises preparing microspheres by spray drying, suspending themicrospheres thus obtained in an aqueous solution containing polyvinylalcohol, and recovering, washing and drying the microspheres, allowsfurther removal of the residual solvent and improves the suspendabilityof microspheres upon administration, thereby leading to accurateadministration of drugs and effective treatment of diseases.

A better understanding of the present invention may be obtained throughthe following examples which are set forth to illustrate, but are not tobe construed as the limit of the present invention.

PREPARATION EXAMPLE 1 Preparation of Microspheres and a Post-ProcessUsing Various Dispersing Agents

PLGA microspheres were prepared using a spray dryer (SODEVA, France)equipped with an ultrasonic nozzle (Sono-Tek, 120 kHz). A biodegradablepolymer, RG503H (Boehringer-Ingelheim, Germany), and a drug, leuprolelinacetate (Polypeptide Laboratories, Denmark), were used. 50 g of RG503Hand 2.5 g of leuprolelin acetate were homogeneously dissolved in 500 mlof glacial acetic acid (Yakuri Pure Chemicals, Japan). The solution wastransported at a flow rate of 3 ml/min using a piston pump. Thetransported solution was sprayed into the spray drier through anultrasonic nozzle installed in an upper part of a sprayer, and driedwith dry air at 200° C. Thereafter, microspheres recovered from acyclone were taken in a certain volume, weighed precisely, added at aconcentration of 50 mg/ml to an aqueous solution containing distilledwater and 1% (w/v) of a dispersing agent, and suspended therein for onehour at room temperature using a magnetic agitator. Dispersing agentsused included polyvinyl alcohol (Sigma, P-8136), polyvinylpyrrolidone(Sigma, PVP-360), human serum albumin (Sigma, A-1654), polyethyleneglycol (Yakuri Pure Chemicals, 28123), Tween 80 (Sigma, P-0343),poloxamer (Sigma, P-1300), sodium carboxymethylcellulose (Sigma,C-5678), gelatin (Sigma, G-6650), glycine (Sigma, G-7126), and mannitol(Sigma, M-8429). The suspension was passed through a vacuum filter.Microspheres thus collected were washed with distilled water two timesand freeze-dried.

TEST EXAMPLE 1 Evaluation of Withdrawal Rates and Syringeability ofMicrospheres

The microspheres prepared in Preparation Example 1 were dispersed in anaqueous solution, and assessed for withdrawal rates into a syringe andsyringeability. Each microsphere formulation was placed into a beaker,and mixed with triple distilled water at a concentration of 50 mg/ml.When microspheres were homogenously dispersed using a magnetic agitator,1 ml of the dispersion was finely withdrawn into a 1-ml syringe fittedwith a 21 gauge needle (n=20). 1 ml of the microsphere suspension in thesyringe was transferred into a 1.5-ml Eppendorff tube and freeze-dried.Microspheres recovered into the Eppendorff tube were assessed for dryweight, and the results are given in Table 1, below.

TABLE 1 Withdrawal rates of microspheres according to the type ofdispersing agents Type of dispersing agents Withdrawal rates (%) RSD (%)Microspheres immediately after 11.4 17.9 spray drying Distilled water22.7 13.4 Polyvinyl alcohol 91.5 4.2 Polyvinylpyrrolidone 51.6 24.4Human serum albumin 70.1 36.4 Polyethylene glycol 66.8 19.0 Tween 8068.8 30.3 Poloxamer 24.2 56.8 Sodium carboxymethylcellulose 60.9 16.9Gelatin 75.7 11.0 Glycine 44.7 39.0 Mannitol 46.5 25.5 Note: RSD(relative standard deviation = [standard deviation of weight ofrecovered microspheres/mean] × 100) indicates the deviation of recoveredmass.

As apparent in Table 1, microspheres immediately after spray drying,which did not undergo a dispersal process, and microspheres thatunderwent a dispersal process in distilled water not containing adispersing agent were found to decrease with respect to withdrawal rateand homogeneity when drawn into a syringe and injected from the syringe.Among several dispersing agents mentioned in the literature studies,polyvinyl alcohol exhibited the highest withdrawal rate, followed bygelatin, human serum albumin and Tween 80. FIG. 1 a shows the withdrawalrates and homogeneity of microspheres, which did not undergo a dispersalprocess or underwent a dispersal process using mannitol and polyvinylalcohol as dispersing agents, when a microsphere suspension was drawninto a syringe and injected from the syringe into a tube. FIG. 1 b showsthe deviation in each tube for the mean withdrawal rate in a syringewhen microspheres did not undergo a dispersal process or underwent adispersal process using polyvinyl alcohol as a dispersing agent. Asshown in FIGS. 1 a and 1 b, microspheres had the highest withdrawal rateand the best homogeneity when undergoing a dispersal process usingpolyvinyl alcohol.

PREPARATION EXAMPLE 2 An Effect of Dispersal Time for Improving theDispersibility of Microspheres

PLGA microspheres were prepared using a spray dryer (SODEVA, France)equipped with an ultrasonic nozzle (Sono-Tek, 120 kHz). A biodegradablepolymer, RG503H, and a drug, leuprolelin acetate, were used. 40 g ofRG503H and 4 g of leuprolelin acetate were homogeneously dissolved in400 ml of glacial acetic acid. The solution was sprayed into a spraydrier through an ultrasonic nozzle at a flow rate of 3 ml/min, and driedwith dry air at 200° C. Thereafter, microspheres recovered from acyclone were taken in a predetermined amount, added at a concentrationof 50 mg/ml to an aqueous solution containing 1% (w/v) polyvinylalcohol, and suspended therein for 1 min, 3 min, 5 min, 10 min, 1 hr, 3hr and 6 hr at 25° C. using a magnetic agitator. The suspension waspassed through a vacuum filter. Microspheres thus collected were washedwith distilled water two times, and freeze-dried.

TEST EXAMPLE 2 Evaluation of Withdrawal Rates and Syringeability ofMicrospheres

The microspheres prepared in Preparation Example 2 were dispersed in anaqueous solution, and assessed for withdrawal rates into a syringe andsyringeability. This test was performed according to the same method asin Test Example 1, and the results are given in Table 2, below.

TABLE 2 Withdrawal rates of microspheres according to dispersal time 1min 3 min 5 min 10 min 1 hr 3 hr 6 hr Withdrawal rates 71.4 80.0 86.189.6 89.4 90.2 90.3 (%) RSD (%) 13.6 11.6 7.8 5.5 5.9 4.9 4.8

As apparent in Table 2, a high withdrawal rate was observed immediatelyafter microspheres were suspended in a polyvinyl alcohol solution. Asthe dispersal time was extended, microspheres exhibited increasedwithdrawal rates and homogeneity when withdrawn into a syringe andinjected from the syringe. However, after a certain suspension time, thewithdrawal rates and homogeneity were maintained at constant levels.

TEST EXAMPLE 3 Measurement of Residual Polyvinyl Alcohol Content

The microspheres prepared in Preparation Example 2 were assessed forresidual polyvinyl alcohol content. This assay was performed bymodifying a method described in Journal of Controlled Release, 82(2002), 105-114, as follows. 2 mg of a formulation, which was accuratelyweighed, were placed into a glass vial, mixed with 400 μl of 0.5 N NaOH,and allowed to react in an oven at 60° C. for 2-3 hrs to be completelydissolved (n=3). The solution, in which microspheres were completelydissolved, was neutralized with 180 μl of 1 N HCR, supplemented with 600μl of 0.65 M boric acid and 100 μl of an I₂/KI (0.05 M/0.15 M) solution,and allowed to react for 20 min. Then, absorbance was measured at 690 nm(UV). The results are given in Table 3, below.

TABLE 3 Residual polyvinyl alcohol amounts according to dispersal time 1min 3 min 5 min 10 min 1 hr 3 hr 6 hr Residual amount 0.02 0.04 0.050.07 0.12 0.22 0.38 (w/w)

As shown in Table 3, the residual amounts of polyvinyl alcohol increasedas the suspension time in the polyvinyl alcohol solution increased.

When the results shown in Tables 2 and 3 were taken together, thedispersibility and withdrawal rates of microspheres improved immediatelyafter microspheres were suspended and dispersed in an aqueous solutionof polyvinyl alcohol, but a dispersal time of about 5 min or longer wasneeded for the optimal effect. In this case, the amount of polyvinylalcohol remaining on the surface of microspheres was more than 0.05 wt%. This is a critical factor in determining the withdrawal rate ofmicrospheres into a syringe.

COMPARATIVE PREPARATION EXAMPLE 1 Preparation of Microspheres byCo-Spraying with Polyvinyl Alcohol

PLGA microspheres were prepared using a spray dryer (SODEVA, France)equipped with an ultrasonic nozzle (Sono-Tek, 120 kHz). A biodegradablepolymer, RG503H, and a drug, leuprolelin acetate, were used. To preparea control microsphere formulation, 1.8 g of RG503H and 0.2 g ofleuprolelin acetate were homogeneously dissolved in 90 ml of glacialacetic acid. The solution was transported at a flow rate of 1.5 ml/minusing a piston pump. The transported solution was sprayed into the spraydrier through an ultrasonic nozzle installed at an upper part of asprayer, and dried with dry air at 200° C. Thereafter, microspheresrecovered from a cyclone were taken in a predetermined amount, added ata concentration of 50 mg/ml to an aqueous solution containing 1% (w/v)polyvinyl alcohol as a dispersing agent, and suspended therein for 1 hrat room temperature using a magnetic agitator. The suspension was passedthrough a vacuum filter. Microspheres thus collected were washed withdistilled water two times and freeze-dried.

A comparative microsphere formulation was prepared by spray dryingmicrospheres and polyvinyl alcohol at the same time in order to comparethe dispersal effect of co-spray dried polyvinyl alcohol with that inthe control formulation. 1.8 g of RG503H and 0.2 g of leuprolelinacetate were homogeneously dissolved in 90 ml of glacial acetic acid.The solution was transported at a flow rate of 1.5 ml/min using a pistonpump. The transported solution was sprayed into the spray drier throughan ultrasonic nozzle installed at an upper part of a sprayer, and driedwith dry air at 200° C, thereby yielding a comparative formulation.

The control and comparative microsphere formulations prepared asdescribed above were individually dispersed in an aqueous solution, andassessed for withdrawal rates into a syringe and syringeability. Thisassay was performed according to the same method as in Test Example 1,and final dry weights of microspheres in tubes were measured. Theresults are given in Table 4, below. The residual polyvinyl alcoholamount of each microsphere formulation was determined according to thesame method as in Test Example 3.

An in vitro release test was carried out, as follows. 10 mg of eachmicrosphere formulation were placed into a 1.5-ml tube, mixed with 1 mlof phosphate buffered saline (PBS), and incubated for 1 hr in anincubator at 37° C. with agitation at 5 rpm using a rotary shaker(SLRM-2M, Seoulin Bioscience). Each reaction solution was centrifuged,and the supernatant was assessed for the amounts of the peptide drugreleased from microspheres using a fluorescence detector (Varian, CaryEclipse; Ex: 280 nm, Em: 350 nm).

TABLE 4 Dispersibility, residual polyvinyl alcohol amounts and initialdrug release of control and comparative microsphere formulationsResidual polyvinyl Initial drug Withdrawal RSD Formulation alcohol (%)release (%) rate (%) (%) Control 0.13 3.1 75.32 5.62 Comparative 0.5215.5 68.18 13.13

As shown in Table 4, compared to the control microsphere formulationundergoing a dispersal process using polyvinyl alcohol, the comparativemicrosphere formulation not undergoing a suspending process had a highresidual polyvinyl alcohol concentration, but exhibited decreasedwithdrawal rate and homogeneity when withdrawn, into a syringe andinjected therefrom, and a high initial drug release rate.

TEST EXAMPLE 4 Evaluation of Residual Solvent Removal Rates According toTime of Dispersal Using Polyvinyl Alcohol

Microspheres were prepared in and recovered from a cyclone according tothe same method as in Preparation Example 2, and suspended in an aqueoussolution of 1% polyvinyl alcohol at a concentration of 50 mg/ml. Whilethe aqueous solution was maintained at 25° C., the removal rates ofresidual acetic acid were measured at given time points. Theconcentrations of residual acetic acid in microspheres were determinedas follows. Microspheres were dissolved in methylene chloride (Junsei,34355-0350), supplemented with an aqueous solution of 0.07% phosphoricacid (Sigma, P-6560), and vigorously mixed therewith. The mixture wascentrifuged to separate a layer of the aqueous phosphoric acid solution.This layer was recovered and assessed for the amount of acetic acidusing HPLC. HPLC was carried out using a C18 column (5 μm, 4.6×250 mm,120 Å). A mixture of 5-50% linear gradient of methanol (J. T. Baker,AH230-4) and 0.07% phosphate buffer (pH 3.0) was used as a mobile phasehaving a flow rate of 1.2 ml/min. Acetic acid was detected at 210 nm(UV), and the results are given in Table 5, below. The residual aceticacid content immediately after microspheres were prepared was 0.8 wt %.

TABLE 5 Residual solvent removal rates according to dispersal time Time(min) Acetic acid removal rates (%) 5 20.0 10 34.2 20 44.0 40 54.5 6061.3 120 73.6 180 87.9

As shown in Table 5, the residual solvent removal rates graduallyincreased with increasing time of suspension of microspheres in anaqueous solution of polyvinyl alcohol to remove the residual solvent.The final residual solvent was maintained at less than 0.1 wt %.

PREPARATION EXAMPLE 3 Preparation of BSA-Loaded Microspheres by SprayDrying of W/O Type Emulsion Containing BSA

0.5 g of bovine serum albumin (BSA) (Sigma, A-7638) was dissolved indistilled water, and homogeneously mixed with a solution prepared bydissolving 9.5 g of RG502H in 95 ml of methylene chloride, therebyyielding a W/O type emulsion. While the emulsion was maintained in anemulsion state using an agitator, it was supplied to a spray drier(Buchi-191) at a flow rate of 3 ml/min. Compressed air was supplied to atwo-fluid nozzle at a flow rate of 450 NL/h to dry sprayed atomizeddroplets using dry air at 80° C. The recovered microspheres weresuspended for 3 hrs in an aqueous solution of 1% polyvinyl alcohol at aconcentration of 50 mg/ml with agitation using a magnetic agitator,washed with distilled water, and freeze-dried. The microspheres thusprepared had a mean particle size of 5.2 μm, and the amount of polyvinylalcohol remaining on the surface of the microspheres was 0.93% (w/w).

PREPARATION EXAMPLE 4 Preparation of BSA-Loaded Microspheres by SprayDrying of S/O Type Emulsion Containing BSA

1 g of BSA was finely pulverized in a mortar, and homogeneously mixedwith a solution prepared by dissolving 9 g of RG502H in 90 ml ofmethylene chloride, thereby yielding an S/O type emulsion. While theemulsion was maintained in an emulsion state using, an agitator, it wassupplied to a spray drier (Buchi-191) at a flow rate of 3 ml/min.Compressed air was supplied to a two-fluid nozzle at a flow rate of 450NL/h to dry sprayed atomized droplets using dry air at 80° C. Therecovered microspheres were suspended for 3 hrs in an aqueous solutionof 1% polyvinyl alcohol at a concentration of 50 mg/ml with agitationusing a magnetic agitator, washed with distilled water, andfreeze-dried. The microspheres thus prepared had a mean particle size of5.8 μm, and the amount of polyvinyl alcohol remaining on the surface ofthe microspheres was 0.85% (w/w).

PREPARATION EXAMPLE 5 Preparation of Leuprolelin-Loaded Microspheres

1 g of leuprolelin acetate and 9 g of RG502H were dissolved in 90 ml ofglacial acetic acid. The solution was supplied to a spray drier(Buchi-191) at a flow rate of 2 ml/min. Compressed air was supplied to atwo-fluid nozzle at a flow rate of 500 NL/h to dry sprayed atomizeddroplets using dry air at 120° C. The recovered microspheres weresuspended for 3 hrs in an aqueous solution of 1% polyvinyl alcohol at aconcentration of 50 mg/ml with agitation using a magnetic agitator,washed with distilled water, and freeze-dried. The microspheres thusprepared had a mean particle size of 5.1 μm, and the amount of polyvinylalcohol remaining on the surface of the microspheres was 0.98% (w/w).

PREPARATION EXAMPLE 6 Preparation of Leuprolelin-Loaded Microspheres

0.4 g of leuprolelin acetate and 9.6 g of R202H were dissolved in 96 mlof glacial acetic acid. The solution was supplied to a spray drier(SODEVA, France) at a flow rate of 3 ml/min, sprayed into a dry chamberusing an ultrasonic nozzle (Sono-Tek, 120 kHz), and dried using dry airat 200° C. The recovered microspheres were suspended for 3 hrs in anaqueous solution of 1% polyvinyl alcohol at a concentration of 50 mg/mlwith agitation using a magnetic agitator, washed with distilled water,and freeze-dried. The microspheres thus prepared had a mean particlesize of 23.4 μm, and the amount of polyvinyl alcohol remaining on thesurface of the microspheres was 0.16% (w/w).

PREPARATION EXAMPLE 7 Preparation of Leuprolelin-Loaded Microspheres

0.5 g of leuprolelin acetate and 9.5 g of R202H were dissolved in 95 mlof glacial acetic acid. The solution was supplied to a spray drier(SODEVA, France) at a flow rate of 3 ml/min, sprayed into a dry chamberusing an ultrasonic nozzle (Sono-Tek, 60 kHz), and dried using dry airat 200° C. The recovered microspheres were suspended for 3 hrs in anaqueous solution of 1% polyvinyl alcohol at a concentration of 50 mg/mlwith agitation using a magnetic agitator, washed with distilled water,and freeze-dried. The microspheres thus prepared had a mean particlesize of 32.6 μm, and the amount of polyvinyl alcohol remaining on thesurface of the microspheres was 0.11% (w/w).

PREPARATION EXAMPLE 8 Preparation of Leuprolelin-Loaded Microspheres

1.4 g of leuprolelin acetate, 0.86 g of RG504H and 7.74 g of R202H weredissolved in 86 ml of glacial acetic acid. The solution was supplied toa spray drier (SODEVA, France) at a flow rate of 3 ml/min, sprayed intoa dry chamber using an ultrasonic nozzle (Sono-Tek, 120 kHz), and driedusing dry air at 200° C. The recovered microspheres were suspended for90 min in an aqueous solution of 1% polyvinyl alcohol at a concentrationof 50 mg/ml with agitation using a magnetic agitator, washed withdistilled water, and freeze-dried.

PREPARATION EXAMPLE 9 Preparation of Octreotide-Loaded Microspheres

0.7 g of octreotide acetate and 9.3 g of RG502H were dissolved in 186 mlof glacial acetic acid. The solution was supplied to a spray drier(SODEVA, France) at a flow rate of 3 ml/min, sprayed into a dry chamberusing an ultrasonic nozzle (Sono-Tek, 120 kHz), and dried using dry airat 200° C. The recovered microspheres were suspended for 1 hr in anaqueous solution of 1% polyvinyl alcohol at a concentration of 50 mg/mlwith agitation using a magnetic agitator, washed with distilled water,and freeze-dried.

TEST EXAMPLE 5

The leuprolelin-loaded microspheres prepared in Preparation Example 8was suspended in a suspension (0.5% (w/w) sodium carboxymethylcellulsoe,5% (w/w) mannitol, 0.1% Tween 80), and subcutaneously injected at asingle dosage of 9 mg/kg (leuprolelin acetate) into male SD rats (n=5,195±20 g). Blood samples were collected from tail veins before drugadministration, 30 min, 1 hr, 3 hr, 6 hr and 1, 2, 4 and 7 days afterdrug administration, and every seven days for a period from day 8 to 90.Rats were challenged with the microspheres at a dosage of 100 μg/kg(based on leuprolelin acetate) 28, 56 and 84 days after drugadministration in order to evaluate the sustained release of the drugfrom the microspheres, and blood samples were collected before drugadministration and 3 and 24 hrs after drug administration. Secondarydrug administration was carried out 90 days after the primary drugadministration, and blood samples were collected as described in theprimary drug administration. The collected blood samples were placedinto 1.5-ml Eppendorff tubes, and centrifuged for 10 min at 4° C. and12,000 rpm. The-obtained sera were stored at −20° C. Serum testosteronelevels were measured using a radio-immunoassay (RIA) Kit (DSL-10-4000,Diagnostic System Laboratories, Inc., Webster, Tex., USA), and theresults are given in FIG. 2. The three challenges, which were performedevery 28 days after drug administration, and the secondary drugadministration, which was performed 90 days after drug administration,resulted in inhibition of the rise of serum testosterone levels. Theseresults indicate that leuprolelin was continuously released over thetest period of 90 days.

TEST EXAMPLE 6

The octreotide-loaded microspheres prepared in Preparation Example 9were suspended in a suspension (0.5% (w/w) sodiumcarboxymethylcellulsoe, 0.6% (w/w) mannitol), and subcutaneouslyinjected at a single dosage of 5 mg/kg (octreotide) to male SD rats(n=6, 195±20 g). Blood samples were collected from tail veins beforedrug administration and 6 hrs and 1, 4, 7, 13, 21 and 31 days after drugadministration. The collected blood samples were placed into 1.5-mlEppendorff tubes, and centrifuged for 10 min at 4° C. and 12,000 rpm.The obtained sera were stored at −20° C. Serum octreotide concentrationswere measured using an enzyme immunoassay (EIA) kit (Bachem, S-1275,Peninsula Laboratories Inc., USA). The results are given in FIG. 3. Asshown in FIG. 3, the octreotide drug was found to be continuouslyreleased for a period of more than two weeks.

INDUSTRIAL APPLICABILITY

As described hereinbefore, the process of preparing microspheresaccording to the present invention allows the preparation of sustainedrelease microspheres not having the problems of conventional preparationmethods of sustained release microspheres, including toxicity due toresidual solvent and poor syringeability. The microspheres preparedaccording to the present invention release an effective concentration ofa drug in a sustained manner for a predetermined period whenadministered to the body, prevent rapid initial drug release, and reducethe required administration frequency of a drug. Thus, the presentmicrospheres are useful in the treatment of diseases.

1. A process of preparing a sustained release microsphere, comprising:spraying a solution, suspension or emulsion containing a biodegradablepolymer, a drug and a solvent into a dry chamber and drying it using dryair to remove the solvent; and dispersing a spray-dried microsphere inan aqueous solution containing polyvinyl alcohol to remove a residualsolvent and improve dispersibility of the microsphere.
 2. The process ofpreparing the sustained release microsphere according to claim 1,wherein the biodegradable polymer is one or more selected from the groupconsisting of polylactide, polyglycolide, poly(lactide-co-glycolide),polyorthoester, polyanhydride, polyhydroxybutyric acid,polycaprolactone, polyalkylcarbonate, and derivatives thereof.
 3. Theprocess of preparing the sustained release microsphere according toclaim 1, wherein the drug is selected from among a peptide and aprotein.
 4. The process of preparing the sustained release microsphereaccording to claim 3, wherein the drug is, selected from amongluteinizing hormone releasing hormone (LHRH) derivatives, somatostatinderivatives, and salts thereof.
 5. The process of preparing thesustained release microsphere according to claim 4, wherein the drug isselected from among leuprolelin, goserelin, triptorelin, octreotide, andsalts thereof.
 6. The process of preparing the sustained releasemicrosphere according to claim 1, wherein in the dispersing in theaqueous solution containing polyvinyl alcohol, polyvinyl alcohol iscoated on the sustained release microsphere in an amount from 0.02% to1.0% by weight.
 7. The process of preparing the sustained releasemicrosphere according to claim 1, wherein in the dispersing in theaqueous solution containing polyvinyl alcohol, the residual solvent inthe sustained release microsphere is additionally removed by more than20%.
 8. A drug-loaded sustained release microsphere, which is preparedby atomizing a solution, suspension or emulsion containing abiodegradable polymer, a drug and a solvent into droplets, drying theatomized droplets to remove the solvent and obtain a dried microsphere,and dispersing the dried microsphere in an aqueous solution containingpolyvinyl alcohol to coat polyvinyl alcohol on the microsphere in anamount from 0.02% to 1.0% by weight.
 9. The drug-loaded sustainedrelease microsphere according to claim 8, wherein the drug is selectedfrom among a peptide and a protein.
 10. The drug-loaded sustainedrelease microsphere according to claim 9, wherein the drug is selectedfrom among luteinizing hormone releasing hormone (LHRH) derivatives,somatostatin derivatives, and salts thereof.
 11. The drug-loadedsustained release microsphere according to claim 10, wherein the drug isselected from among leuprolelin, goserelin, triptorelin, octreotide, andsalts thereof.